Induced pluripotent stem cells (also known as iPSCs) are a type of pluripotent stem cell generated directly from adult cells. The iPSC technology was pioneered by Japanese Shinya Yamanaka, demonstrating in 2006 that the introduction of four specific genes (Oct4, Sox2, cMyc and Nanog) encoding transcription factors could convert adult cells into pluripotent stem cells.
Currently, pluripotent stem cells hold great promise in the field of regenerative medicine. Its objectives are to repair tissues altered by accident, diseases or aging. This represents a new therapeutic field with tremendous medical impact because it offers the possibility to treat and cure diseases currently without adequate treatment. Regenerative medicine applies to most medical domains and constitutes one of the most promising developments of the biotechnology industry. Ischemic, degenerative and/or ageing-associated diseases are the major causes of mortality in the population of developed countries.
It is widely recognized that stem cells could be a source of cells for cellular therapy for these diseases because of their proliferative and differentiating capacity. However, medical applications of embryonic stem cells are hampered by immunological and ethical concerns.
These two obstacles are set aside by the iPSC (induced Pluripotent Stem Cell) technology.
iPSCs offer autologous cell sources for replacement cell therapy, to replace or regenerate tissues by autologous transplantation. Moreover, patient-specific iPSCs can serve as in vitro models for disease mechanism modeling and drug screening.
The original set of reprogramming factors (also called Yamanaka factors) comprises the genes Oct4, Sox2, cMyc and Nanog. More recently, some specific combinations including three of these factors were reported to also generate iPSC.
However, reprogramming of iPSC using these factors only occurs in a very small percentage of transfected cells demonstrating that the therapeutic use of iPSCs has to face many barriers among which safety, regulatory issues, financial viability, low reprogramming efficiency and an uncertain stability of derived iPSC.
Various solutions to the problem of low reprogramming efficiency have been proposed, based on the regulation of the expression (overexpression or inhibition) of other genes, in addition to at least some of the Yamanaka factors. However, the methods proposed in the prior art for increasing reprogramming efficiency generally result in DNA lesions and chromosomal aberrations, due to the concomitant induction of alterations in DNA repair machinery. As a result, while increasing reprogramming efficiency, proposed methods are not suitable for the preparation of high numbers of clinically useful iPSCs.
For instance, the original Yamanaka factors include c-myc, an oncogene that is known to alter DNA repair machinery and to result in DNA lesions and chromosomal aberrations. While this factor is in fact not necessary to induce iPSCs, using only the other Yamanaka factors results in decreased reprogramming efficiency.
Recent studies have documented the role of p53 in stem cell (SC) homeostasis and in pluripotency induction. p53 not only ensures genomic integrity after genotoxic insults but also controls SC proliferation and differentiation. Additionally, p53 provides an effective barrier to the generation of iPSCs from terminally differentiated cells. If wild-type (wt) p53 has inhibitory effects, some p53 mutants display completely opposite effects (Sarig et al, 2010). A recent genome wide study demonstrated that p53 regulates approximately 3600 genes in mouse ES cells (Li et al, 2012). Out of these, about 2000 genes are positively regulated while 1600 are repressed. Positively regulated genes are enriched in genes responsible for differentiation while negatively regulated genes, in maintaining ES cell status. p53 represses key regulators of ES phenotype like Oct 4, Nanog, Sox2, c-myc (Li et al, 2012; Lin et al, 2005). Consequently, recent reports showed that p53 is an obstacle to cellular reprogramming through p21 signalling pathway (Hong et al, 2009).
It was found that the depletion of p53 significantly increased efficacy of cell reprogramming, concomitantly providing iPSC generated using only two factors (Sox2 and Oct 4) of the Yamanaka cocktail (Kawamura et al, 2009). Takenaka et al. also disclose that the inhibition of p53 allows obtaining iPSC from CD34+ monocytes (2010).
However, generating iPSC by p53 depletion or expression of mutated p53 proteins carries great risk due to the fact that these cells present tumor like features and develop malignant tumors when injected in mice. The permanent suppression of p53 may thus lower the quality of iPSCs, in particular by accumulating DNA lesions and chromosomal aberrations during iPSC derivation and maintenance (Tapia & Scholer, 2010). So the use in cell therapy of iPSC obtained by the depletion of p53 genes or the expression of mutated p53 proteins is hazardous and unsure.
Otherwise, the TP53 gene encodes at least twelve different physiological isoforms [TAp53 (α, β and γ), Δ40p53 (α, β and γ), Δ133p53 (α, β and γ) and Δ160p53 (α, β and γ)] (Bourdon, 2007) via several mechanisms: use of alternative promoters (the TA and Δ133 isoforms), alternative intron splicing (intron 2: Δ40 isoforms and intron 9: α, β and γ isoforms) and alternative translational initiation sites (Δ40 isoforms and Δ160 isoforms). A scheme summarizing the features of isoforms α, β and γ of TAp53, Δ40p53, and Δ133p53 is presented in FIG. 1. The TAp53α isoform is the best described and classically mentioned in the literature as p53. Basically, p53 isoforms can be divided in two groups: long isoforms that contain the transactivation domain (TA and Δ40) and short isoforms without transactivation domain (Δ133 and Δ160). Furthermore, the β and γ isoforms do not contain the canonical C-terminal oligomerization domain, but an additional domain with unknown function(s) to date (Khoury and Bourdon, 2011). p53 isoforms can regulate p53 transcriptional activity and have different outcomes on cell fate by regulating cell cycle progression, programmed cell death, replicative senescence, viral replication, cell differentiation and angiogenesis (Aoubala et al, 2011; Bourdon et al, 2005; Hofstetter et al, 2012; Nutthasirikul et al, 2013; Terrier et al, 2012).
WO 2012/044979 discloses the use of Δ133p53α isoform for increasing reprogramming efficiency and thus iPSCs induction in the presence of at least one reprogramming factor selected from Yamanaka factors. However, this document contains no data showing that iPSC obtained with the method disclosed are genetically stable and do not have DNA damages or alterations in DNA repair machinery. Consequently, there is still a need for methods allowing efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) that are free of genetic damages and have normal somatic cells functions and in particular an unaltered DNA repair machinery, thus guaranteeing their genetic stability and their possible clinical use.